Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics

Peña, Jennifer M; Manning, Brendan J; Li, Xiang; Fiore, Elizabeth S; Carlson, Leah; Shytle, Kristen; Nguyen, Peter P; Azmi, Ishara F.; Larsen, Alex; Wilson, Mary K; Singh, Subha; DeMeo, Marisa C; Ramesh, Pradeep; Boisvert, Heike; Blake, William J.

doi:10.1016/j.jmoldx.2023.03.009

Cited by 3 publications

(3 citation statements)

References 26 publications

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“… 720 Furthermore, the Real-Time-Multiplexed SHERLOCK assay (real-time SLK) developed by Pena et al enables simultaneous detection of multiple samples with sensitivity down to 5 copies/μL and a time to result of under 30 min. 721 The CRISPR-based detection is not limited to viruses, but has also been expanded to the detection of other microorganisms, including bacteria, fungi and parasites. 722 – 724 This highlights its potential as an important next-generation detection technology.…”

Section: Conclusion and Perspectivementioning

confidence: 99%

Nanotechnology’s frontier in combatting infectious and inflammatory diseases: prevention and treatment

Huang,

Guo,

et al. 2024

Sig Transduct Target Ther

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Inflammation-associated diseases encompass a range of infectious diseases and non-infectious inflammatory diseases, which continuously pose one of the most serious threats to human health, attributed to factors such as the emergence of new pathogens, increasing drug resistance, changes in living environments and lifestyles, and the aging population. Despite rapid advancements in mechanistic research and drug development for these diseases, current treatments often have limited efficacy and notable side effects, necessitating the development of more effective and targeted anti-inflammatory therapies. In recent years, the rapid development of nanotechnology has provided crucial technological support for the prevention, treatment, and detection of inflammation-associated diseases. Various types of nanoparticles (NPs) play significant roles, serving as vaccine vehicles to enhance immunogenicity and as drug carriers to improve targeting and bioavailability. NPs can also directly combat pathogens and inflammation. In addition, nanotechnology has facilitated the development of biosensors for pathogen detection and imaging techniques for inflammatory diseases. This review categorizes and characterizes different types of NPs, summarizes their applications in the prevention, treatment, and detection of infectious and inflammatory diseases. It also discusses the challenges associated with clinical translation in this field and explores the latest developments and prospects. In conclusion, nanotechnology opens up new possibilities for the comprehensive management of infectious and inflammatory diseases.

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Section: Conclusion and Perspectivementioning

confidence: 99%

Nanotechnology’s frontier in combatting infectious and inflammatory diseases: prevention and treatment

Huang,

Guo,

et al. 2024

Sig Transduct Target Ther

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show abstract

“…Recently, the combination of Cas12-subtypes [ 36 ] and Cas13-subtypes [ 37 ] was widely adopted for collateral cleavage-based one-pot multiplexing. The collateral cleavage substrate is different between Cas12 (ssDNA) and Cas13 (ssRNA), and the sequence of the substrate depends on the Cas subtype.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, multiplex PCR-like detection systems can be realized by classifying Cas types, reporter sequences, and probe types for different targets. However, this sequence specificity of ssDNA substance has only been confirmed in two subtypes of Cas12 [ 36 ] and four subtypes of Cas13 [ 37 ], severely limiting the amount of detectable target genes. Overall, previous attempts at collateral cleavage-based multiplexing still exhibited limitations in simultaneously achieving comprehensive genotyping and simple operation.…”

Section: Introductionmentioning

confidence: 99%

Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing

Shigemori,

Fujita,

Tamiya

et al. 2024

Micromachines

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DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12–crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12–crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.

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